Abacavir is a
nucleoside reverse transcriptase inhibitor marketed since 1999 for the treatment of
infection with the human immunodeficiency virus type 1 (HIV). Despite its clinical efficacy,
abacavir administration has been associated with serious and sometimes fatal toxic events.
Abacavir has been reported to undergo bioactivation in vitro, yielding reactive species that bind covalently to
human serum albumin, but the haptenation mechanism and its significance to the toxic events induced by this
anti-HIV drug have yet to be elucidated.
Abacavir is extensively metabolized in the liver, resulting in inactive
glucuronide and carboxylate metabolites. The metabolism of
abacavir to the carboxylate involves a two-step oxidation via an unconjugated
aldehyde, which under
dehydrogenase activity isomerizes to a conjugated
aldehyde. Concurrently with metabolic oxidation, the two putative
aldehyde metabolites may be trapped by nucleophilic side groups in
proteins yielding covalent adducts, which can be at the onset of the toxic events associated with
abacavir. To gain insight into the role of
aldehyde metabolites in
abacavir-induced toxicity and with the ultimate goal of preparing reliable and fully characterized prospective
biomarkers of exposure to the drug, we synthesized the two putative
abacavir aldehyde metabolites and investigated their reaction with the α-amino group of
valine. The resulting adducts were subsequently stabilized by reduction with
sodium cyanoborohydride and derivatized with
phenyl isothiocyanate, leading in both instances to the formation of the same
phenylthiohydantoin, which was fully characterized by NMR and MS. These results suggest that the unconjugated
aldehyde, initially formed in vivo, rapidly isomerizes to the thermodynamically more stable conjugated
aldehyde, which is the electrophilic intermediate mainly involved in reaction with bionucleophiles. Moreover, we demonstrated that the reaction of the conjugated
aldehyde with
nitrogen bionucleophiles occurs exclusively via
Schiff base formation, whereas soft
sulfur nucleophiles react by Michael-type 1,4-addition to the α,β-unsaturated system. The synthetic
phenylthiohydantoin adduct was subsequently used as standard for LC-ESI-MS monitoring of N-terminal
valine adduct formation, upon modification of human
hemoglobin in vitro with the conjugated
abacavir aldehyde, followed by reduction and Edman degradation. The same postmodification strategy was applied to investigate the products formed by incubation of
abacavir with rat liver cytosol, followed by trapping with ethyl valinate. In both instances, the major adduct detected corresponded to the synthetic
phenylthiohydantoin standard. These results suggest that
abacavir metabolism to the carboxylate(s) via
aldehyde intermediate(s) could be
a factor in the toxic events elicited by
abacavir administration. Furthermore, the availability of a reliable and fully characterized synthetic standard of the
abacavir adduct with the N-terminal
valine of
hemoglobin and its easy detection in the model
hemoglobin modifications support the usefulness of this adduct as a prospective
biomarker of
abacavir toxicity in humans.